Levonorgestrel (LNG) is a synthetic progestin widely used as a contraceptive for humans ^[1]. However,LNG has deleterious reproductive effects including catamenia,increasing rate of galactophore cancer,etc. ^[2]. Methylprednisolone (MP) is a synthetic glucocorticoid used as an immunosuppressive effector or as a veterinary drug. The excessive application of MP is associated with significant toxicity such as diabetes,hypertension, metabolic disorders,etc.^{[3, 4]}. Because most LNG and MP residues will be eventually discarded to environmental water system and function as environmental hormones,displaying potential risk to humans and ecosystems,there is an urgent need for fast,sensitive and simultaneous detection of these compounds in water samples.

Some analytical methods for LNG and MP detection have been reported,including high performance liquid chromatography (HPLC) ^{[5, 6]},electrochemical detection ^[7],liquid chromatography-mass spectrometry (LC-MS) ^[8],thin-layer chromatography ^[9] and gas chromatography-mass spectrometry (GC-MS) ^[10],etc. However in the methods above,expensive instruments,skilled personnel,and complex and time-consuming sample treatments are involved. Immunochromatographic assays (ICA) have been gaining increasing attention for rapid and on-site detection of proteins,pesticides,drugs and toxins in different matrices ^{[11, 12, 13, 14, 15, 16, 17]}. To our knowledge,there is no report of ICA for simultaneous detection of two environmental hormones in water samples. The aim of this study is to develop a rapid immunochromatographic assay for simultaneous detection of LNG and MP in water samples.

The polyclonal antibodies against LNG and MP (e.g.,pAb_LNGand pAb_MP) were produced by immunizing New Zealand rabbits using LNG-bovine serum albumin (BSA) and MP-BSA as immunogens in our laboratory. The LNG (or MP) was chemically modified,so that the LNG (or MP) derivative contained a space arm with a carboxylic group at the end. The LNG (or MP) derivative was coupled to carrier protein (BSA) to form the immunogen which was injected into rabbits for the production of pAb ^{[18, 19]}. Colloidal gold with an average diameter of 18.6 nm were prepared according to the procedures in literature ^[13]. Briefly,100 mL of 0.01% HAuCl₄ solution in pure water was heated to boiling and 2.5 mL of 1% trisodium citrate solution was added while stirring. After the color changed from dark blue to wine red,the solution was boiled for another 5 min,then cooled to room temperature and stored at 4℃ with 0.05% sodium azide. The colloidal gold-labeled pAbs (e.g.,AupAb_LNGand Au-pAb_MP) were prepared as follows. 5 mL of pAb_LNG (or pAb_MP) at the optimum concentration was incubated with 50 mL of colloidal gold solution (pH 9) at 4℃ overnight. After adding 5 mL 5% BSA solution for blocking,the mixtures werecentrifuged (13,000 rpm) at room temperature for 40 min. The precipitates were resuspended with 5 mL of 0.02 mol/L sucrose solution containing 1% BSA and 0.05% sodium azide and stored at 4℃ for use.

Assembly of the test strips was similar to the procedure in literature ^[13]. The test strip consists of an absorbent pad,a NC membrane,conjugate,and a sample pad. The PVC sheet was used as the backing plate of the test strip. LNG-ovalbumin (OVA) and MP-OVA as test lines and goat anti-rabbit IgG (GaRIgG) as control line were separately immobilized on NC membrane. The distance between lines was about 5 mm. The conjugate pad was prepared by dispensing Au-pAb_LNGand Au-pAb_MP on a glass fiber,and dried at 37℃ for 60 min. The conjugate pad was pasted on the PVC sheet by overlapping about 2 mm with the NC membrane. Sample pad and absorbent pad were pasted on the two sides of the strip by overlapping by 2 mm. The assembled plate was cut to 4 mm width of strips. The strips were stored in a desiccator at 4℃.

LNG and MP standard solutions at 0,0.1,1,10,100 and 1000 ng/mL were prepared by diluting LNG and MP stock solution (0.5 mg/mL in methanol) with pure water. Two types of water samples including pond water and lake water were collected and spiked with LNG and MP at 0,1,5,10 and 20 ng/mL. The spiked water samples were tested by ICA three times. Standard or sample (100μL) was dripped into the sample holder of the test strip cell. While the solution moved to the adsorbent pad,red color on the test line could be observed by the naked eye.Theoverallassaytimeofthe ICA was about 10 min.

The assay is based on the competitive reaction of coating antigen (test line) and target analyte in standard (or sample) for limited antibodies on Au-pAb. The principle and result judgments of ICA for simultaneous detection of LNG and MP are illustrated in Fig. 1. When the standard (or sample) solution moved forward on the absorbent pad,Au-pAb_LNGand Au-pAb_MPon conjugate pad was dissolved and moved together with solution. If there was no LNG and MP in solution (the negative test for both LNG and MP),AupAb_LNG and Au-pAb_MPwould be captured by LNG-OVA and MPOVA coated on test lines. Excess Au-pAb_LNGand Au-pAb_MPwould move continuously to the control line and be captured by GaRIgG. Two red bands on test lines and one red band on control line would appeared due to the accumulation of red colored Au-pAb. In contrast,if there was LNG or MP in solution (positive testing for single or dual compounds),Au-pAb_LNG or Au-pAb_MPwould be firstly reacted with LNG or MP,leading to less conjugates being captured by LNG-OVA or MP-OVA on test lines. The color intensity of relevant test lines was visually weaker than that of the control line. The more LNG and MP presented in the solution,the weaker the test lines appeared. Thus,the degree of gold-color on the test lines was the reverse of the concentration of LNG or MP. If no line presented in the control region,the test was considered to be invalid ^{[20, 21]}.

Fig. 1

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Fig. 1. Principle of result judgment on one-step strip assay for simultaneous detection of LNG and MP. (A) Negative for two compounds; (B) LNG positive and MP negative; (C) LNG negative and MP positive; (D) positive for two compounds; and (E1) and (E2) invalidation of the strip.

The colloidal gold particles were homogeneous in size ranging 15-20 nm with a mean diameter of 18.6 nm. This is within the typical range of colloidal gold used in ICA. The Au-pAbs were measured by TEM as shown in Fig. 2.

Fig. 2

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Fig. 2. Transmission electron micrograph of the Au (A),Au-pAbLNG(B) and Au-pAbMP(C).

For simultaneous detection of LNG and MP by ICA strips,various conditions were tested,and the optimal conditions were obtained as follows: 0.5 mg/mL of LNG-OVA and 0.2 mg/mL of MP-OVA were used for test lines. The optimum concentration of pAb_LNG and pAb_MPfor preparing Au-pAb conjugates were 60μg/mL and 50μg/mL,respectively; 200μL of the mixture of Au-pAb_LNG (100μL) and Au-pAb_MP(100μL) diluted with 200mL 8.0% (w/v) sucrose solution containing 1.0% (w/v) BSA and 0.05% (w/v) sodium azide were dispensed on the conjugate pad.

LNG and MP standards ranging 0-1000 ng/mL were applied to ICA strips and the results were illustrated in Fig. 3. It was seen that the same intensity of red color appeared on control lines on different strips,indicating validity of the assay. In addition,as the concentration of LNG or MP (single or dual) increased,the intensity of red color on test lines decreased. For LNG testing,the intensity of the red color on the test line at 1 ng/mL of LNG was significantly weaker than that at zero concentration,and the red line completely disappeared at 10 ng/mL of LNG. Thus,10 ng/mL was considered the visual detection limit of the ICA for LNG. Similarly,the detection limit of the ICA for MP was also found to be 10 ng/mL. As ICA is commonly used as a semi-quantitative analytical method,its sensitivity was 2-5 times lower than that of corresponding ELISA; this phenomenon was also observed for the proposed ICA ^{[18, 22]}. The sensitivity of the LNG detection achieved by ELISA was found to be 3.3 ng/mL ^[22],but the proposed method can be used for rapid and on-site simultaneous detection of LNG and MP. Moreover,the specific binding between one analyte and its corresponding Au-pAb was not affected by the presence of another analyte,demonstrated the feasibility of the ICA strip for simultaneously detecting two target analytes. To test the selectivity further,the standard solutions of seven compounds including five structurally relative compounds (estriol,17α-estradiol,17β-estradiol,estrone,prednisolone) and two other compounds (diethylstilbestrol,clenbuterol) in the concentration range of 0-10,000 ng/mL were applied to ICA strips. It was found that there was no cross-reactivity of the proposed ICA with these compounds,revealing the high selectivity of the ICA.

Fig. 3

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Fig. 3. ICA strips for simultaneous detection of LNG and MG standard solutions (upper line is the control line; middle and bottom lines are the MP and LNG test lines, respectively). (A) LNG/MP concentrations (from left to right): 0/0; 0.1/0; 1/0; 10/0; 100/0 and 1000/0 ng/mL; (B) LNG/MP concentrations (from left to right): 0/0; 0/0.1; 0/1; 0/ 10; 0/100 and 0/1000 ng/mL; and (C) LNG/MP concentrations (from left to right): 0/0; 0.1/0.1; 1/1; 10/10; 100/100 and 1000/1000 ng/mL.

The spiked water samples were applied to ICA procedures. In comparison,the spiked water samples were also detected by enzyme-linked immunosorbent assay (ELISA). The results obtained by ICA and ELISA for LNG and MP detection were summarized in Table 1. For nonspiking water samples,negative results (－) were obtained by ICA; for water samples spiked with LNG and MP both at concentration of 5 ng/mL,the weak positive results (±) were obtained; when LNG and MP were spiked at 10 and 20 ng/mL,the positive results (+) were achieved. The results from ICA for the detection of LNG and MP were also agreement with those from ELISA, suggesting that the proposed ICA is able to semi-quantitatively detect LNG and MP in water samples. The stability of the assay was evaluated and it demonstrated that the strips could be stored at 4℃ for 10 weeks without significant loss of activity.

Table 1

Table 1 Comparison of ELISA and ICA for detecting LNG/MP in water samples (n= 3)

Table 1 Comparison of ELISA and ICA for detecting LNG/MP in water samples (n= 3)

A one-step ICA for simultaneous detection of LNG and MP in water samples has been developed. The visual detection limits of the ICA for LNG and MP in water samples were 10 ng/mL. The assay time was 10 min without sample pretreatment. To our knowledge, this is the first ICA for simultaneous detection of LNG and MP in water samples. The proposed ICA provides a feasible tool for rapid and simultaneous detection of LNG and MP residues in water samples on site.

H.F. Chang and J.Q. Wang contributed equally to this work. The authors are grateful for financial support from the National Natural Science Foundation of China (No. 21175097) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.